We evaluated the potential clinical utility of a reverse transcription-polymerase chain reaction (RT-PCR) for neonatal enterovirus meningitis, comparing the results with viral culture and white blood cell (WBC) counts in the cerebrospinal fluid (CSF). Of the 41 cases of enteroviral infection, 31 cases (76%) were finally diagnosed as meningitis by either viral culture, CSF WBC count or RT-PCR. Of those with culture positive and negative, the RT-PCR positive rates were 10 (100%) and 6/13 (46%), respectively. Of those with and without WBC increase in the CSF, the RT-PCR positive rates were 18/20 (90%) and 5/14 (36%), respectively. There was one RT-PCR positive case (1/6, 17%) among those with culture negative and no WBC increase in the CSF. RT-PCR performed on CSF is a sensitive and specific method for the diagnosis of neonatal enterovirus meningitis.